This application is a renewal of an ongoing Program Project in which five investigators with different but complementary expertise have worked together to understand two fundamentally important levels of TCR signaling: 1) how the TCR regulates the tyrosine kinases (SFKs, Syk kinases and Tec kinases) that control critical downstream tyrosine phosphorylation; and, 2) how Ras activation, a critical downstream signaling pathway, is regulated by Ras guanine nucleotide exchange factors (GEFs) whose activities themselves are coupled to TCR signaling via the set of substrates of the kinases controlled by the TCR. Our collaborative studies have resulted in considerable progress. In this renewal, our overall goal is to capitalize on our current progress and bring together approaches from structural biology, proteomics, immunology, and computational biology to understand TCR signaling. In project #1, we will study the distinct features of the T cell-expressed SFKs, Syk and Tec kinases that make these more suitable for antigen receptor signaling in T cells than their counterparts in B cells. We hypothesize that the characteristics of Lck and Fyn, ZAP-70 and Itk and their signaling regulators have been optimized in T cells to establish signaling circuitry that serves to maintain a basal signaling state that is resistant to perturbations by non-agonist peptides and also establishes a sensitive threshold for optimal recognition and response to agonist pMHC. In project #2, we hope to understand how basal and TCR-induced RasGEF signaling regulates the primed but controlled state of peripheral T cells while allowing for efficient T cell activation. We hypothesize that the SOS1 and RasGRP1 RasGEFs have evolved to be regulated in distinct manners to allow for non-redundant Ras signals in T cells that establish the homeostasis/activation balance.